A Novel ESIPT Phthalimide-Based Fluorescent Probe for Quantitative Detection of H2O2.

Hydrogen peroxide (H2O2) is a majority reactive oxygen species (ROS) and acts as an essential role in pathological and physiological processes. Therefore, the development of quantitative detection of methods for H2O2 is necessary. Here, we constructed of a novel simple fluorescence probe for detection of H2O2 based on the excited-state intramolecular proton transfer process. The probe utilized a phthalimide derivative as the fluorophore and selected phenylboronic acid as the recognition site for H2O2. In response to H2O2, the probe exhibited 63-fold fluorescence intensity enhancement, a low detection limit (8.4 × 10-8 M), and large Stokes shift (111 nm). In addition, the probe displayed high selectivity for H2O2 over other ROS. Moreover, the probe was successfully employed for imaging of H2O2 in living cells.

Introduction

As a major member of the reactive oxygen species (ROS) family, hydrogen peroxide (H2O2) plays a key role in host defense, proliferation, cell growth, and signaling pathways in the physiological process.[1−4] However, excessive H2O2 generation results in certain diseases, such as Parkinson’s disease, cardiovascular disease, Alzheimer’s disease, cancer, and inflammatory disease.[5−8] On the other hand, H2O2 is also widely applied to industry and day-to-day life including bleaching and disinfection. However, the significant concentration of H2O2 in wastewater can be involved in severe oxidative damage in organisms.[9] Therefore, the development of quantitative monitoring methods for H2O2 is very meaningful.

Currently, there is a lot of analytical approaches for detection of H2O2, such as the electrochemical method,[10] colorimetric method,[11] mass spectrometry,[12] high-performance liquid chromatography.[13] Though the above methods work well for detection of H2O2, most of them require destruction of cells or tissues and complicated preparation. Thus, they are not suitable for application to the live cells. As powerful tools for monitoring analytes, fluorescent probes are desirable for detection of analyte biological samples because of their fast response, high selectivity, good compatibility for biosamples, and nondestructive analysis.[14−23] To date, many fluorescent probes for monitoring H2O2 have been constructed, such as metal complexes,[24] catechol,[25] and boronate ester.[26] However, some of these fluorescent probes are not satisfactory for detection of H2O2 because of their detection sensitivity.[27] In addition, some of them have small Stokes shifts (<70 nm)[28,29] that is a significant limitation toward biological applications due to the interference from autofluorescence. The large Stokes shifts can minimize self-quenching and thereby improve the detection accuracy.[30,31] Thus, it is a great necessity to develop a simple fluorescent probe with large Stokes shifts that avoided self-quenching for quantitative detection of H2O2.

Phthalimide derivatives with a typical excited-state intramolecular proton transfer (ESIPT) process have been recognized as ideal fluorophores in the design of probes owing to the large-fluorescence Stokes shift, high quantum yield, good photostability, and biocompatibility.[32] Herein, we presented a phthalimide-boronate as the ESIPT-process fluorescence probe for detection of H2O2. The probe used 3-hydroxyphthalimide as the fluorophore and boronate ester as the H2O2 recognition group. In this probe, the ESIPT process would be blocked and thus could result in fluorescence to be efficiently quenched due to the protection of the OH moiety with benzyl boronic pinacol ester. However, oxidation of the phenylboronic pinacol ester mediated by H2O2 would release the OH moiety and would recover its ESIPT process in the molecule, which caused a fluorescence increase. Notably, the probe displayed a large Stokes shift toward H2O2. Furthermore, the probe exhibited high selectivity and sensitivity for detection of H2O2 over other ROS under mild conditions. Moreover, the probe was successfully applied for fluorescence H2O2 imaging in living cells with satisfactory results, suggesting its value for practical application.

Results and Discussion

Optical Properties

The spectroscopic analysis of the probe BBD was carried out in PBS (pH 7.4, 50 mM, 50% CH3CN as a cosolvent). In the presented absorption spectra (Figure S1), no obvious absorption was observed at 401 nm, but upon the addition of H2O2, one could find prominent changes in absorption at 401 nm, which suggested the H2O2-triggered cleavage reaction between H2O2 and probe BBD. Then the fluorescence spectra of the probe for H2O2 also were investigated in the absence and presence of H2O2. As expected, the probe BBD displayed weak fluorescence due to the EISPT process being blocked from the boronate ester moiety. With the introduction of H2O2, the solution of probe BBD appeared to have a strong green fluorescence at 512 nm (Figure ). When the solution of probe BBD was incubated with different concentrations of H2O2, the fluorescence spectra of probe BBD exhibited changes. The fluorescence intensity of probe BBD increased with the gradual addition of H2O2, and a plateau appeared when 4.5 equiv of H2O2 was added. Meanwhile, a wonderful linearity was exhibited based on the plot between the fluorescence intensities at 512 nm and H2O2 concentrations (0.0–45.0 μM), and the detection limit was estimated to be 8.4 × 10–8 M, suggesting that the probe BBD has sensitivity for detection of H2O2 (Table S1). Furthermore, the probe BBD displayed a large Stokes shift (111 nm) toward H2O2. The good linear relationship and low detection limit demonstrated that the probe BBD was suitable for quantitative detection of H2O2 in aqueous media.

Figure 1

(a) Fluorescence spectra of probe BBD (10 μM) with the gradual addition of H2O2 in PBS (pH 7.4, 50 mM, 50% CH3CN as a cosolvent); (b) linear relationship between the fluorescence intensities at 512 nm and H2O2 concentrations (0.0–45.0 μM). Excited at 401 nm.

Selectivity Studies

To determine its selectivity for H2O2, fluorescence responses of probe BBD to other relevant species were investigated including reactive oxygen species (H2O2, BOO–, –O2, ·OH, ONOO–,ClO–, NO, and HNO) and ions (NO3–, NO2–, Fe3+, Zn2+, Cu2+, Ni2+, Co2+, Mg2+, and Ca2+). As shown in Figure , only the existence of H2O2 could induce a significant fluorescence enhancement of probe BBD. However, the other species resulted in negligible fluorescence changes. Those results suggested that the probe BBD could be used for high-selectivity detection of H2O2.

Figure 2

Response times of probe BBD (10.0 μM) at 512 nm in the absence (b) and presence (a) of 45 μM H2O2 in PBS (pH 7.4, 50 mM, 50% CH3CN as a cosolvent). Excited at 401 nm.

Kinetic Studies

The dynamic behavior of probe BBD was recorded in the absence or presence of H2O2. As depicted in Figure , the probe BBD displayed negligible fluorescence changes at 512 nm emission along with the time, which illustrated that the probe BBD was stable in solution. Upon addition of 4.5 equiv of H2O2, the fluorescence enhancement was observed and the maximum fluorescence value appeared at 26 min. These results indicated that probe BBD has a fast response for H2O2.

Figure 3

Emission of probe BBD (10 μM) at 512 nm with (black) or without (red) H2O2 (45 μM). Excitation at 401 nm.

Effect of pH

In order to evaluate the practicability of probe BBD under physiological conditions, the effect of pH on the fluorescence response of probe BBD in the absence and presence of H2O2 in various pH were examined (Figure ). It could be seen that fluorescence intensities of probe BBD scarcely changed in the pH range from 2.00 to 11.00. In the presence of H2O2, negligible fluorescence changes were observed at pH 2.00–5.00. However, there were notable changes for fluorescence of probe BBD in the pH range from 6.00 to 11.00, which indicated that the probe BBD could be used for monitoring H2O2 under physiological conditions.[33]

Figure 4

Fluorescence spectra of probe BBD (10 μM) at 512 nm after incubation with different species.

Response Mechanism

To further confirm the sensing mechanism of probe BBD for H2O2, the isolated product of probe BBD reacting with H2O2 was demonstrated by 1H NMR spectra (Figure S5). From the 1H NMR spectrum, it could be seen that the reaction product of probe BBD with H2O2 was in good agreement with that of compound 1. On the other hand, when H2O2 was added into the probe BBD, a mass peak at 277.1183 m/z was observed, which was consistent with that of compound 1 ([M + H]+ = 277.2878) (Figure S6). Thus, according to these results, the sensing mechanism of the probe BBD with H2O2 was confirmed in Scheme .

Scheme 1

Proposed Mechanism of Probe BBD to H2O2

Cellular Imaging

To inquire into practical application of probe BBD, we first investigated its cytotoxicity to HeLa cells with MTT assays. As shown in Figure S7, the survival of cells was more than 85% when the cells were treated with the probe BBD (10 μM), which illustrated that probe BBD has low cytotoxicity in living cells. Then the H2O2 imaging of probe BBD was carried out in living cells (Figure ). When cells were pretreated with probe BBD (10 μM), weak fluorescence was exhibited inside the cells. After addition of H2O2 for 30 min, a strong green fluorescence signal appeared. The results suggested that probe BBD was able to image H2O2 in the living cells.

Figure 5

Fluorescence images of HeLa cells. (a) Bright-field and (b) fluorescence images of HeLa cells incubated with probe BBD (10 μM) for 30 min. (c) Bright-field and (d) fluorescence images of HeLa cells preincubated with probe BBD for 30 min and then stimulated with H2O2 for 30 min.

Conclusions

In summary, we have developed a simple novel phthalimide-based ESIPT-process fluorescent probe BBD for detection of H2O2. The probe is able to qualitatively detect H2O2 with an excellent linearity in a concentration range from 0.0 to 45.0 μM through H2O2-induced hydrolysis of aromatic boronic ester. Furthermore, the probe exhibits a large Stokes shift, high selectivity, and sensitive response to H2O2, which are beneficial for biological application. Importantly, the probe has potential to detect H2O2 in living cells.

Experimental Section

Materials and Instruments

4-Hydroxyisobenzofuran-1,3-dione was purchased from Sinopharm Chemical Reagent Company. Other reagents were obtained from Heowns Biochemical Technology Company. 4-Hydroxy-2-(2-morpholinoethyl)isoindoline-1,3-dione was synthesized according to the literature.[33] A Bruker AVB-500 spectrometer was used to obtain NMR (1H and 13C NMR) spectra. An Agilent 6530 Accurate-Mass Q-TOF LC/MS was used to record electrospray ionization mass spectra. A Hitachi F-7000 spectrophotometer and UV2600 UV–vis spectrophotometer were used to collect fluorescence spectra and absorbance spectra, respectively.

Synthesis of Probe BBD

4-Hydroxy-2-(2-morpholinoethyl)isoindoline-1,3-dione (0.2763 g, 1 mmol), 4-(bromomethyl)benzeneboronic acid pinacol ester (0.3553 g, 1.2 mmol), K2CO3 (0.1658 g, 1.2 mmol), and CH3CN (10 mL) were added in a glass tube. After stirring and refluxing overnight, the reaction mixture was cooled and then evaporated. The crude solid was separated by silica column chromatography using (ethyl acetate/petroleum ether = 1:8), and probe BBD was obtained with 62% yield (Scheme ). 1H NMR (500 MHz,CDCl3) δ (ppm): 7.35 (d, 2H, J = 6.5 Hz), 7.57 (d, 1H, J = 6.5 H), 7.50 (d, 2H, J = 6.5 Hz), 7.44 (d, 1H, J = 7.0 Hz), 7.16 (d, 1H, J = 8.5 Hz), 5.40 (s, 2H), 3.83 (d, 2H), 3.69 (t, 4H), 2.66 (t, 2H), 2.56 (t, 2H), 1.37 (s, 12H); 13C NMR (125 MHz, CDCl3) δ (ppm): 168.0, 166.8, 155.7, 139.0, 135.2, 134.5, 126.0, 120.1, 119.5, 118.1, 115.8, 83.9, 70.9, 56.2, 53.5, 34.8, 24.9; HRMS calculated for 492.3717 [M + H]+, found 493.2500.

Scheme 2

Synthetic Route of Probe BBD

Measurement Procedure

The stock solution of probe BBD (1 × 10–3 M) was prepared in CH3CN. The stock solutions of various relevant analytes were dissolved in PBS buffer (50 mmol/L, pH 7.4). The absorption and fluorescence spectra were obtained in CH3CN-PBS (1:1 v/v). The fluorescence spectra were measured with excitation of 400 nm.

Cell Culture

The HeLa cells were grown in a 96 well plate at 37 °C for 24 h. Then the culture medium of the cells was pretreated with 10 μM probe BBD and incubated for 30 min. After washing three times with PBS, the cells were incubated with H2O2 for 30 min at 37 °C. The fluorescence images were studied by an inverted NIKON Eclipse Ti-S fluorescence microscope.